To investigate the sperm ultrastructure and kinematic characteristics of Pareuchiloglanis anteanalis and Pareuchiloglanis gongshanensis, a systematic comparative analysis was conducted using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and a computer-assisted sperm analysis (CASA) system. The results revealed that while spermatozoa of both species consist of an acrosome-less head, a midpiece, and a single flagellum, they exhibit significant divergence in morphological configuration and swimming patterns. Spermatozoa of P. anteanalis were relatively longer, characterized by a slender head (length-to-width ratio: 1.51), a midpiece containing four umbrella-shaped and tightly arranged mitochondria, and a flagellum with symmetrical lateral fins. In contrast, P. gongshanensis spermatozoa were shorter, featuring a nearly spherical head (ratio: 1.08), a midpiece with 4–5 smaller mitochondria, and a shorter flagellum with more developed lateral fins. CASA measurements indicated no significant differences in average path velocity (VAP), straight-line velocity (VSL), and curvilinear velocity (VCL) between the two species. However, P. anteanalis sperm exhibited significantly higher straightness (STR) and beat-cross frequency (BCF) (P < 0.05), resulting in a more linear swimming trajectory. Conversely, P. gongshanensis sperm showed a significantly greater amplitude of lateral head displacement (ALH) (P < 0.05) and a more curved movement path. This study suggests that the morphological and kinematic traits of P. anteanalis sperm are highly consistent with the typical phenotype of "introsperm" (internally fertilizing sperm), whereas those of P. gongshanensis reflect an evolutionary adaptation to external fertilization (aquasperm).